HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) A correction has been published Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Martineau, A. R. Articles by Wilkinson, R. J. Search for Related Content PubMed PubMed Citation Articles by Martineau, A. R. Articles by Wilkinson, R. J.

IFN-- and TNF-Independent Vitamin D-Inducible Human Suppression of Mycobacteria: The Role of Cathelicidin LL-37¹

Adrian R. Martineau^*,,, Katalin A. Wilkinson^*,, Sandra M. Newton^*, R. Andres Floto, Anthony W. Norman^¶, Keira Skolimowska^*,, Robert N. Davidson^||, Ole E. Sørensen^#, Beate Kampmann^*,, Christopher J. Griffiths and Robert J. Wilkinson^2,*,,||,**

^* Wellcome Trust Center for Research in Clinical Tropical Medicine, Division of Medicine, Wright Fleming Institute, Imperial College London, United Kingdom; Centre for Health Sciences, Queen Mary’s School of Medicine and Dentistry, Barts and The London, London, United Kingdom; Institute of Infectious Diseases and Molecular Medicine, Faculty of Health Sciences, University of Cape Town, Observatory, South Africa; Department of Medicine, Cambridge Institute for Medical Research, Cambridge, United Kingdom; ^¶ Department of Biochemistry, University of California, Riverside, CA 92521; ^|| North West London Hospitals National Health Service Trust, Northwick Park Hospital, Harrow, United Kingdom; ^# Section of Clinical and Experimental Infection Medicine, Department of Clinical Sciences, Lund University, Biomedical Center, Lund, Sweden; and ^** Department of Medicine, Faculty of Health Sciences, University of Cape Town, Observatory, South Africa

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Vitamin D deficiency is associated with susceptibility to tuberculosis, and its biologically active metabolite, 1,25 dihydroxyvitamin D₃ (1,25(OH)₂D₃), has pleiotropic immune effects. The mechanisms by which 1,25(OH)₂D₃ protects against tuberculosis are incompletely understood. 1,25(OH)₂D₃ reduced the growth of mycobacteria in infected human PBMC cultures in a dose-dependent fashion. Coculture with agonists or antagonists of the membrane or nuclear vitamin D receptors indicated that these effects were primarily mediated by the nuclear vitamin D receptors. 1,25(OH)₂D₃ reduced transcription and secretion of protective IFN-, IL-12p40, and TNF in infected PBMC and macrophages, indicating that 1,25(OH)₂D₃ does not mediate protection via these cytokines. Although NOS2A was up-regulated by 1,25(OH)₂D₃, inhibition of NO formation marginally affected the suppressive effect of 1,25(OH)₂D₃ on bacillus Calmette Guérin in infected cells. By contrast, 1,25(OH)₂D₃ strongly up-regulated the cathelicidin hCAP-18 gene, and some hCAP-18 polypeptide colocalized with CD14 in 1,25(OH)₂D₃ stimulated PBMC, although no detectable LL-37 peptide was found in supernatants from similar 1,25(OH)₂D₃-stimulated PBMC cultures. A total of 200 µg/ml of the active peptide LL-37, in turn, reduced the growth of Mycobacterium tuberculosis in culture by 75.7%. These findings suggest that vitamin D contributes to protection against TB by "nonclassical" mechanisms that include the induction of antimicrobial peptides.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Tuberculosis (TB)³ is a globally important cause of death (1). Although effective chemopreventive and chemotherapeutic regimes exist, their implementation is not straightforward because of the need for prolonged (6–9 mo) treatment courses. Interruption of such treatment also contributes to the growing problem of drug resistant disease (2). Therefore, there is a need for novel interventions that might help prevent and treat this disease.

It is recognized that the majority of people infected with TB do not develop disease. Instead, a lifelong state of concomitant immunity referred to as latent TB infection develops following primary infection. Perturbation of concomitant immunity by immunosuppression can rapidly lead to the development of reactivation or postprimary TB many years after the initial infection. The CD4 T cell depletion that characterizes HIV infection is by far the strongest associate of such reactivation, although only 7–12% of the global TB burden can be attributed to this cause (3). Immunosuppression by corticosteroid therapy and anti-TNF agents and via rare genetic defects in the IL-12- and IFN--driven type 1 cytokine pathway also predispose to active TB (4, 5, 6, 7, 8). However, the overall contribution of these recognized risk factors to global TB burden is most likely small, and the mechanisms that lead to the breakdown of immunity to TB in most humans remain to be discovered.

A factor that associates epidemiologically with the reactivation of TB is migration, best documented in those moving from endemic environments to more developed areas of the world where TB incidence in indigenous people is very low (9, 10, 11). In 2005 in the United States, the risk of TB in persons born in Asia was 19.6 times greater than in the white population (12). There is some evidence that the incidence of TB in such immigrants is greater than in the country of origin (9). Factors that may change as a consequence of migration are diet and sunlight exposure and thereby the level of vitamin D (13). An association between vitamin D deficiency and the risk of TB in foreign borns in London, U.K., exists (14, 15, 16), and other findings corroborate the idea that vitamin D is protective (17, 18, 19). Historically, vitamin D was used in the treatment of TB until the advent of modern chemotherapy (20).

Vitamin D has no direct antimycobacterial action, but its active metabolite, 1,25 dihydroxyvitamin D₃ (1,25(OH)₂D₃), modulates the immune response to Mycobacterium tuberculosis (MTB). Expression of macrophage 25-hydroxyvitamin D 1-hydroxylase (1-hydroxylase) has been shown recently to be up-regulated by ligation of macrophage TLRs by MTB Ags (21). This enzyme metabolizes 25-hydroxyvitamin D₃ (25(OH)D₃) to 1,25(OH)₂D₃. IFN- secreted by type 1 T cells potentiates this effect by up-regulating 1-hydroxylase (22) and inhibiting induction of 25(OH)D 24-hydroxylase (23), a key enzyme in 1,25(OH)₂D₃ inactivation. 1,25(OH)₂D₃ induces antimycobacterial activity in vitro in both monocytes (MN) (24) and macrophages (17). Several mechanisms of action have been proposed. Exogenous 1,25(OH)₂D₃ induces a superoxide burst (25) and enhances phagolysosome fusion (26) in MTB-infected macrophages; both phenomena are mediated by PI3K, suggesting that this response is initiated by ligation of a membrane vitamin D receptor (VDR) (27). 1,25(OH)₂D₃ also modulates immune responses by binding the nuclear VDR, where it up-regulates protective innate host responses, including the induction of NO synthase, NOS2A (28). There is also evidence that LRG-47 (an IFN--inducible 47-kDa vacuolar guanosine triphosphatase) is required to protect mice against TB (29). There has been no investigation of the single human homolog of LRG-47 (IRGC) in human TB.

The purpose of this study was to investigate in vitro mechanisms by which 1,25(OH)₂D₃ might increase the ability of PBMC from sensitized human donors to resist mycobacteria. We found that 1,25(OH)₂D₃ suppressed both bacillus Calmette Guérin (BCG) and MTB in infected cell cultures. These effects were primarily mediated by the nuclear VDR. 1,25(OH)₂D₃ negatively regulated the transcription and secretion of IFN-, IL-12p40, and TNF in MTB- and BCG-infected PBMC and macrophage cultures, indicating that vitamin D does not mediate protection via these cytokines. Although the NOS2A gene was up-regulated by 1,25(OH)₂D₃, inhibition of the formation of reactive nitrogen intermediate (RNI) only had a slight effect on the suppressive effect of 1,25(OH)₂D₃ on BCG in PBMC cultures. By contrast 1,25(OH)₂D₃ very strongly up-regulated (and MTB down-regulated) the cathelicidin gene, hCAP18. Intracellular hCAP18 protein was also increased by 1,25(OH)₂D₃ and synthetic LL-37, the antimicrobial peptide derived from hCAP18, reduced the growth of MTB in culture by up to 75.7%. Our findings indicate that vitamin D mediates protection against TB by "nonclassical" mechanisms, including the induction of antimicrobial peptides.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Coculture of PBMC and mycobacteria

PBMC were isolated from the buffy coats of healthy purified protein derivative-reactive blood donors over Ficoll as described previously (30). PBMC (5 x 10⁵) were plated in triplicate on 48-well plates in RPMI 1640/10% FCS. Infection and subsequent handling of cultures was according to techniques described previously (31). MN preparations were obtained by adherence, and periodic assessment by FACS found them to be 82 ± 4% CD14 and 76 ± 7% CD11b positive. 1,25(OH)₂D₃ and its analogs were dissolved in ethanol (0.1–0.2% final concentration in culture). Vehicle control experiments indicated that this concentration had no affect on the growth of bacilli or on the gene expression and cytokine secretion of eukaryotic cells. PBMC were preincubated with 1,25(OH)₂D₃ and its analogs for 72 h, then infected, then culture supernatants for cytokine analysis were harvested at 24 h for TNF and 96 h for IFN- and IL-12p40 for subsequent analysis by ELISA. L-NG-monomethylarginine (L-NMMA) (Sigma-Aldrich) was used in some cell cultures at a final concentration of 5 mM (a 4.35 molar excess over the concentration of L-arginine in RPMI 1640). To investigate the stability of cathelicidin LL-37 in cell culture, PBMC were isolated as above and plated in duplicate on 48-well plates in RPMI 1640/10% FCS spiked with 1 µg/ml synthetic LL-37 (PANATecs). Supernatants were aspirated after 0, 24, 48, 72, and 96 h of culture, and the concentration of cathelicidin LL-37 was determined by ELISA.

Recombinant mycobacteria

MTB-BCG and MTB H37Rv transformed with a replicating vector (pSMT1) containing the luciferase gene of Vibrio harveyi under the control of a constitutive promoter (Hsp60, Rv0440c) were prepared as described previously (32). Frozen aliquots of bacilli were grown to mid-log phase in Middlebrook 7H9 supplemented with 10% Albumin Dextrose Catalase (Difco) and 15 µg/ml hygromycin. Periodic relative luciferase units (RLU)-CFU determinations were conducted to ensure plasmid stability. Knowledge of the CFU:RLU ratio was used to infect with equal numbers of log-phase bacilli corresponding to a multiplicity of infection (MOI) mononuclear phagocyte:bacillus of 1:1 into cell culture. Following lysis of eukaryotic cells in 1 ml of H₂O, the luminescence of duplicate 100 µl aliquots of suspension of bacilli was determined by measuring the area under the curve decay in luminescence over 20 s in the presence of excess n-decyl aldehyde substrate (Sigma-Aldrich) in a luminometer (Berthold Technologies) as described previously (33). CFU enumeration was performed by plating serial dilutions of bacilli on 7H11 agar in triplicate.

Incubation of MTB under iron-restricted conditions

One potentially important difference between the ionic environment in 7H9 medium and the phagolysosome is free iron concentration: 1.5 x 10^–4 M in 7H9 broth but estimated to be 10^–8 M within the phagolysosome (34). Iron-free 7H9 medium was prepared from its constituent ingredients (omitting ferric ammonium citrate) in bottles washed with 6 M HCl. Iron-depleted stocks of H37Rv lux were prepared by inoculation of iron-replete stock into iron-free medium to yield a final iron concentration of 2 µM, grown to log phase, and frozen after addition of an equal concentration of 30% glycerol (final concentration 1 µM Fe in 15% glycerol). Inocula from these iron-depleted stocks were then added to iron-free medium with or without 5 µg/ml LL-37 at 1/100 dilution to attain 10^–8 M final Fe concentration.

RNA extraction and quantitative RT-PCR

For RNA extraction, 5 x 10⁶ PBMC or MN in 2 ml were set up in 6-well plates. In addition, both mononuclear phagocytes (isolated by adherence as previously described (35)) and PBMC from the same donors were studied. In these cultures, the infecting dose of MTB was normalized to average MN count (10% total PBMC) such that the MOI was 0.1:1 for PBMC and 1:1 for mononuclear phagocytes. Culture supernatant was aspirated and the cell monolayer immediately lysed using the RNEasy extraction kit (Qiagen). RNA was reverse transcribed using the Quantitect reverse transcription kit (Qiagen) that includes a DNase digest step. As the gene for LRG-47 has a single exon, we confirmed the completion of DNase digestion by PCR from partially processed samples that had not undergone the reverse transcriptase step. cDNA was used in quantitative PCR for IFN-, TNF, IL-12p40, NOS2A, LRG-47 (IRGC), LL-37, and -actin on the ABI Prism 7000 platform. Primers and probes were obtained as predeveloped assay reagents (Applied Biosystems) with the exception of IRGC (XM_293893) for which the primers and probe sequences were as follows: forward primer, 5'-TCCCACTTTTCAAATGTGGTGTT-3'; reverse primer 5'-TCAGGTAGTTCTCCAGGGTTGTG-3'; and probe 5'-6-FAM-ACCTGCCTGGCACAGGGTCTGC-3'-TAMRA.

Each reaction was multiplexed by, and normalized to the -actin content and fold induction over unstimulated samples was calculated by the C_T method as described previously (user bulletin no. 2, available for download from www.appliedbiosystems.com).

ELISA

Supernatants were analyzed for the presence of TNF and IL-12p40 by ELISA using Ab pairs from R&D Systems, and IFN- was assayed using an Ab pair from BD Pharmingen (catalog nos. 554548 and 554550). The sensitivity of these assays was between 10 and 77 pg/ml. LL-37 was assayed using a kit from Hycult Biotechnology for LL-37 (HK321) whose sensitivity was 1 ng/ml.

FACS analysis

Adherent MN were cocultured in the presence of varying concentrations of 1,25(OH)₂D₃ in 0.1% ethanol for 72 h. Staining for surface HLA-DR expression was performed according to standard protocols using PE-conjugated anti-HLA-DR and PE-conjugated mouse IgG1 as isotype control (Serotec). Stained cells were analyzed on a FACSCalibur flow cytometer equipped with CellQuest software.

Immunostaining for hCAP-18

Rabbit hCAP-18 polyclonal antiserum (36) and a PE-conjugated mouse mAb to CD14 (BD Biosciences) were used to detect hCAP-18 in mononuclear phagocytes. The rabbit polyclonal Ab is highly specific for hCAP-18, as demonstrated by immunoblotting of both neutrophil homogenate and plasma (36), and it reacts with both the LL-37 and the cathelin domain of the hCAP-18 molecule (37). PBMC from a healthy laboratory donor were isolated above and cultured in quadruplicate with 10^–6 M 1,25(OH)₂D₃ or 0.1% ethanol control for 72 h on 12-mm poly-L-lysine-coated coverslips (VWR) in 24-well plates. Cells were fixed in 3.7% paraformaldehyde, washed three times in PBS 10 mM HEPES, and incubated for 15 min at room temperature with permeabilization solution (PS) containing 10% calf serum, 0.05% saponin, 10 mM glycine and 10 mM HEPES. Cells were then incubated at room temperature for 30 min in PS containing 2% MACS FcR-blocking reagent (Miltenyi Biotec) with or without 1% hCAP-18 antiserum. Following three additional washes in PS, all cells were incubated at room temperature for 30 min in PS containing 1% goat anti-rabbit Alexa Fluor 644 (Molecular Probes) 17% CD14 PE Ab (BD Biosciences) and 2% MACS FcR-blocking reagent. After three additional washes in PS, coverslips were mounted onto glass slides and dried overnight at 4°C before confocal microscopy. Experiments substituting preimmune rabbit IgG for anti-hCAP-18 antiserum (to control for nonspecific binding of primary Ab) and staining in the absence of anti-hCAP-18 antiserum (to control for nonspecific binding of secondary Ab) were also performed.

Statistical analysis

Normally distributed variables were analyzed by Student’s paired or unpaired t test as appropriate. Correlation was performed by calculation of the Pearson or Spearman according to the normality of variables. RNA fold induction values were normalized by log₁₀ transformation. Significance was inferred when p values were <0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
1,25(OH)₂D₃ suppresses the growth of mycobacteria in cells

The ability of 1,25(OH)₂D₃ to suppress the growth of BCG-lux in the PBMC of 10 healthy donors was tested. PBMC (5 x 10⁵) were inoculated with a fixed RLU content of BCG lux (t = 0) corresponding to a MOI of 0.1 CFU:cell. The growth of BCG over the following 96 h in the presence of varying amounts of 1,25(OH)₂D₃ was determined by dividing the luminescence at 96 h by that at t = 0 to give a luminescence ratio (LR) for each donor. 1,25(OH)₂D₃ was associated with a dose-dependent reduction in LR that became statistically significant at 10^–8 M (Fig. 1A). The effect was dependent on the presence of cells because 1,25(OH)₂D₃ had no affect on the LR of BCG-lux that had been cultured in tissue culture medium alone.

View larger version (10K): [in this window] [in a new window]   FIGURE 1. Ability of 1,25(OH)2D3 to suppress the growth of BCG-lux. A, PBMC of 10 healthy donors were inoculated with a fixed RLU content of BCG-lux. The growth of BCG over the following 96 h in the presence of varying amounts of 1,25(OH)2D3 was determined by dividing the luminescence at 96 h by that at t = 0 to give a LR for each donor. 1,25(OH)2D3 was associated with a dose-dependent reduction in luminescence (•) that became statistically significant at 10–8 M. This effect was dependent on the presence of cells because 1,25(OH)2D3 had no affect in their absence (). B, PBMC of 10 donors were cultured with BCG-lux and MTB-lux in the presence or absence of 10–6 M 1,25(OH)2D3. 1,25(OH)2D3 induced a fall in the growth of BCG-lux from 1.74 x 106 to 9.66 x 105 RLU/ml and a fall in MTB-lux from 1.05 x 106 to 5.81 x 105 RLU/ml (p = 0.002 and <0.0001, respectively). C, PBMC of 6 donors were cultured with MTB-lux for 96 h in the presence or absence of 10–6 M 1,25(OH)2D3. Significant suppression of the CFU of MTB was seen in all donors with the mean decreasing from 8.83 x 104 to 5.51 x 104 CFU/ml (p = 0.031). A similar decrease in luminescence in the same cultures was also observed (p = 0.063). Error bars, SE.

The protective effect of 1,25(OH)₂D₃ against mycobacteria is predominantly mediated via nuclear signaling

1,25(OH)₂D₃ can ligate a membrane-bound VDR (VDR_mem) to initiate rapid effects or a nuclear receptor (VDR_nuc) to modulate downstream gene transcription (27). We investigated the pathway responsible for the suppression of mycobacteria using conformationally locked analogs of 1,25(OH)₂D₃ that selectively agonize or antagonize these receptors (Table I). All analogs were dissolved in ethanol, which was present in cell culture at final concentration of 0.1–0.2%. Neither vehicle control nor any analog had an effect on the luminescence of BCG-lux cultured in medium alone (data not shown).

View larger version (18K): [in this window] [in a new window]   FIGURE 2. The protective effect of 1,25(OH)2D3 against mycobacteria requires nuclear signaling. A, The selective nuclear VDR agonist V (•) was associated with dose-dependent suppression of BCG-lux in PBMC culture that was evident at a concentration as low as 10–9 M, whereas the membrane-specific agonist JN () had no affect. B, In four donors, 10–8 M 1,25(OH)2D3 significantly suppressed BCG-lux (p < 0.001). Increasing concentrations of the VDRmem antagonist HL were unable to reverse this suppression. C, In six additional donors, MK (a partial antagonist of the nuclear VDR) had moderate agonist effects at 10–7 M. The antagonist activity of MK was revealed by complete reversal of the suppressive effect of 10–8 M 1,25(OH)2D3 (p = 0.016 by comparison with cultures that contained 1,25(OH)2D3 alone). Error bars show SE.

Cytokine secretion and HLA-DR expression in cells cocultured with BCG-lux and 1,25(OH)₂D₃

There is clear evidence that containment of MTB requires IL-12-mediated differentiation of type 1 lymphocytes producing IFN- that thereby activates mononuclear phagocytes to increase their ability to present Ag via up-regulation of HLA-DR and to produce a variety of mediators, including TNF. We were interested to determine whether the 1,25(OH)₂D₃-mediated in vitro suppression of BCG-lux that we had observed could be related to these factors. We therefore assayed the BCG-lux-induced secretion of IFN- and IL-12p40 at 96 h and TNF at 24 h in the presence of varying concentrations of 1,25(OH)₂D₃ in culture supernatants from 13 donors. In a subset of 4 donors, we also determined the effect of 1,25(OH)₂D₃ on HLA-DR expression (in the absence of BCG) by FACS analysis. There was a 1,25(OH)₂D₃ dose-dependent reduction in IFN-, IL-12p40, and TNF secretion that became significant for all three mediators at 10^–9 M 1,25(OH)₂D₃ (p 0.008; Fig. 3, A–C). There was also a clear trend toward decreased expression of HLA-DR, although in the smaller numbers of donors tested, this did not attain significance (p = 0.125; Fig. 3D).

View larger version (8K): [in this window] [in a new window]   FIGURE 3. Cytokine secretion and HLA-DR expression in cells cocultured with BCG-lux and 1,25(OH)2D3. The BCG-lux induced secretion of IFN- and IL-12p40 at 96 h, and TNF at 24 h in the presence of varying concentrations of 1,25(OH)2D3 in culture supernatants from 13 donors was assayed. A–C, There was dose-dependent 1,25(OH)2D3 suppression of IFN-, IL-12p40, and TNF secretion that became significant for all three cytokines at 10–9 M (p 0.008). D, In a subset of four donors, the effect of 1,25(OH)2D3 on HLA-DR expression was determined. There was a trend toward decreased expression of HLA-DR (p = 0.125). Error bars, SE.

MTB- and 1,25(OH)₂D₃-mediated regulation of genes involved in protection against MTB

In addition to IL-12, IFN-, and TNF, there is also evidence that NO and LRG-47 (an IFN--inducible 47-kDa vacuolar guanosine triphosphatase) are required to protect mice against TB (29, 38). There has been no investigation of the single human homolog of LRG-47 (IRGC) in human TB. To further investigate NO and IRGC and to generalize the cytokine results we had obtained in 1,25(OH)₂D₃ and BCG-lux-stimulated cultures, we therefore investigated the regulatory effects of both MTB and 1,25(OH)₂D₃ on these genes.

The PBMC and MN of 10 donors were used in these experiments. An initial sample of RNA was extracted from both cell types to determine the effect of preincubation with 10^–6 M 1,25(OH)₂D₃ or solvent (0.1% ethanol) for 72 h before infection with MTB. After this 72-h preincubation, time point 0 RNA samples were extracted for both cell types. These samples served as controls for the RNA extracted from 6 to 24 h. MTB-stimulated samples in the continued presence or absence of 1,25(OH)₂D₃. Two broad patterns of regulation were observed: genes up-regulated strongly by MTB and down-regulated by 1,25(OH)₂D₃ (IL-12p40, IFN-, and TNF; Fig. 4, A–C) and genes whose expression was variably affected by MTB and 1,25(OH)₂D₃ (IRGC and NOS2A; Fig. 4, D and E).

View larger version (19K): [in this window] [in a new window]   FIGURE 4. The PBMC and MN of 10 donors were stimulated with MTB in the presence or absence of 1,25(OH)2D3 (D) for 6 or 24 h, followed by RNA extraction and quantitative RT-PCR. A, 1,25(OH)2D3 decreased constitutive IL-12p40 in MN over the initial 72 h (p = 0.03). MTB strongly up-regulated (21- to 117-fold) IL-12p40 in MN (p 0.011). This was reversed (a 17,660-fold decrease at t = 6 h) by the continued presence of 1,25(OH)2D3 (p < 0.0001). B, 1,25(OH)2D3 decreased constitutive IFN- expression in both MN and PBMC during the 72-h preinfection culture (p 0.03). MTB strongly up-regulated (55- to 724-fold) IFN- in both cell types at both time points (p 0.0004). This up-regulation was reversed (130- to 1,778-fold decrease) by the continued presence of 1,25(OH)2D3 (p 0.0001). C, MTB also up-regulated TNF (9- to 112-fold, p 0.01) in both cell types at both time points. This up-regulation was reversed (22- to 369-fold decrease) by the presence of 1,25(OH)2D3 (p < 0.0001). D, 1,25(OH)2D3 moderately increased the constitutive expression of IRGC in MN (2.1-fold, p = 0.017) and conversely decreased by 2.9-fold IRGC expression in MTB-stimulated PBMC at 6 h (p = 0.04). No other effect attained statistical significance. E, 1,25(OH)2D3 increased the constitutive expression of NOS2A 3.9-fold in PBMC (p = 0.0008). In MTB-stimulated PBMC at 24 h, the mean expression of NOS2A was 5.25-fold increased (p = 0.005). Error bars, SE. The y-axis is the mean log10 fold induction.

The effects of both 1,25(OH)₂D₃ and MTB on IRGC were moderate and variable between donors (Fig. 4D). The only statistically significant effects on IRGC expression were a moderate (2.1-fold, p = 0.017) increase in constitutive expression in MN and conversely a 2.9-fold 1,25(OH)₂D₃-associated decrease in MTB-stimulated PBMC at 6 h. 1,25(OH)₂D₃ increased the constitutive expression of NOS2A 3.9-fold in PBMC (p = 0.0008; Fig. 4E). NOS2A was not regulated by MTB, but at 24 h, its mean expression in MTB-stimulated PBMC was also 5.25-fold increased (p = 0.005) by 1,25(OH)₂D₃.

1,25(OH)₂D₃-mediated suppression of BCG-lux is only slightly impaired by inhibition of NO

Because 1,25(OH)₂D₃ increased the expression of NOS2A, we determined whether inhibition of the formation of RNI by L-NMMA would reverse the 1,25(OH)₂D₃-mediated decrease in luminescence. The PBMC of four donors were therefore set up with BCG-lux in the presence or L-NMMA with or without 10^–8 M 1,25(OH)₂D₃. L-NMMA has no effect on the luminescence of BCG-lux grown in medium. In PBMC culture, L-NMMA increased (by an average of 58%) the 96-h LR, suggesting that RNI do play a role in controlling BCG-lux under the conditions of this assay (Fig. 5). 1,25(OH)₂D₃ decreased the luminescence as observed previously. However, the addition of L-NMMA to the 1,25(OH)₂D₃-stimulated culture resulted only in a modest (25%) increase in RLU per milliliter. Taken together, these results suggest that the major component of 1,25(OH)₂D₃-mediated suppression of mycobacteria is by a mechanism other than the generation of NO.

View larger version (11K): [in this window] [in a new window]   FIGURE 5. 1,25(OH)2D3-mediated suppression of BCG-lux is only slightly impaired by inhibition of NO. PBMC from four donors were set up with BCG-lux in the presence of L-NMMA (5 mM) with or without 10–8 M 1,25(OH)2D3. L-NMMA increased (by an average of 58%) the 96-h LR. 1,25(OH)2D3 decreased the luminescence by 41%. However, the addition of L-NMMA to the 1,25(OH)2D3-stimulated cultures resulted only in a modest (25%) increase in RLU per milliliter. Error bars, SE.

1,25(OH)₂D₃ up-regulates cathelicidin hCAP18, and the active peptide LL-37 decreases the growth of MTB

1,25(OH)₂D₃ is known to induce the expression of the hCAP-18 gene that encodes the antimicrobial peptide LL-37 (39). Therefore, we were interested to determine the effect of MTB on the expression of this gene and whether LL-37 had activity against MTB. PBMC and MN of 10 donors were set up, and RNA was extracted for quantitative RT-PCR as described above. 1,25(OH)₂D₃ increased constitutive hCAP-18 expression in both MN and PBMC over the initial 72 h by between 50- and 206-fold (p 0.002; Fig. 6A). In MN, MTB down-regulated (4.59- to 13.7-fold) LL-37 at 6 and 24 h (p 0.015). 1,25(OH)₂D₃ (10^–6 M) completely reversed (527- to 774-fold increase) this MTB mediated suppression of hCAP-18 (p 0.0001). The presence of hCAP-18 peptide in 1,25(OH)₂D₃-treated mononuclear phagocytes was confirmed by immunohistochemical staining; some peptide colocalized with CD14 (Fig. 6B). No staining for hCAP-18 was observed in the absence of primary Ab (data not shown) or when preimmune rabbit IgG was substituted for primary Ab (Fig. 6B), indicating that staining was specific for hCAP-18.

View larger version (54K): [in this window] [in a new window]   FIGURE 6. Regulation of the cathelicidin hCAP18 by 1,25(OH)2D3 and its effect on protein production. A, PBMC and MN of 10 donors were set up and RNA extracted as described previously. 1,25(OH)2D3 increased constitutive cathelicidin gene expression 50- to 206-fold in both MN and PBMC over the initial 72 h (p 0.002). In MN, MTB down-regulated (4.59- to 13.7-fold) cathelicidin at 6 and 24 h (p 0.015). 1,25(OH)2D3 (10–6 M) completely reversed (527- to 774-fold increase) this MTB-mediated suppression of cathelicidin (p 0.0001). B, Confocal microscopy of PBMC cultured in the presence or absence of 10–6 M 1,25(OH)2D3 for 72 h. Immunostaining for hCAP-18 is shown in red and CD14 in green. Some hCAP-18 colocalized with CD14 in 1,25(OH)2D3-stimulated cells. No staining for hCAP-18 was observed when preimmune rabbit IgG was substituted for anti-hCAP-18 antiserum, indicating that binding of primary Ab was specific. Optical sectioning at 1 µM intervals revealed positive staining for hCAP-18 in a granular distribution, with more diffuse staining for CD14. These granular areas of positive hCAP-18 staining were not continuous between sections, indicating that some hCAP-18 is located in the intracytoplasmic compartment.

To assay the effect of synthetic LL-37 peptide on the growth of MTB, six replicates of MTB were set up in broth containing LL-37 and cultured for 96 h. There was a dose-dependent reduction in the CFU of MTB that was maximal (75.7% reduction) at 200 µg/ml (p = 0.04; Fig. 7A). The microbicidal effects of LL-37 depend on the ionic environment (40). One potentially important difference between the ionic environment in 7H9 medium and the phagolysosome is free iron concentration: 1.5 x 10^–4 M in 7H9 broth but estimated to be 10^–8 M within the phagolysosome (34). MTB requires free iron for electron transport and the generation of ATP. Therefore, we investigated the effects of a physiological low-dose (5 µg/ml) synthetic LL-37 on MTB under iron-limiting conditions. At 96 h, the suppressive effects of both iron depletion in the presence or absence of LL-37 were moderate and did not achieve significance. However, when iron-depleted cultures were prolonged to 192 h, the CFU recovered from cultures containing 5 µg/ml LL-37 were 1.8-fold reduced (2.42 x 10⁶ vs 4.42 x 10⁶, p < 0.0001) when compared with cultures without LL-37. By contrast, no effect of 5 µg/ml LL-37 on MTB CFU was observed in the presence of 150 µM iron (Fig. 7B).

View larger version (8K): [in this window] [in a new window]   FIGURE 7. Effect of synthetic cathelicidin LL-37 on the growth of MTB. A, LL-37 led to a dose-dependent reduction in the growth of MTB in broth culture that became significant at 2 µg/ml (p = 0.039) and was maximal (75.7% reduction) at 200 µg/ml. The mean of six replicates is shown with or without SE. B, The effect of low-dose (5 µg/ml) LL-37 on MTB under iron-limiting conditions. At 192 h, the CFU recovered from cultures containing LL-37 and 10–8 M free iron were 2-fold reduced (2.42 x 106 vs 4.96 x 106, p < 0.0001) when compared with cultures that contained LL-37 in the presence of 2 µM iron. Mean of 12 replicates ± SE.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

When modeling the replication of mycobacteria in humans, we and others (30, 35, 41) have classically infected mononuclear phagocytes with washing off of nonphagocytozed bacilli after an interval (the "CFU assay"). In this way, the intracellular replication of bacteria in variously differentiated cells can be assessed. However, we did not in these experiments restrict our consideration of the action of 1,25(OH)₂D₃ to mononuclear phagocytes. In addition, MTB is able to replicate extracellularly in vivo. Lastly, the CFU assay is cumbersome for the high-throughput analyses that we desired. We therefore adopted coculture of light emitting BCG and MTB with PBMC to study the many of these effects. Although our experience is that tissue culture medium poorly supports the growth of mycobacteria, we acknowledge that, as in tissue, they may have been able to metabolize and replicate extracellularly. However, the presence of cells consistently reduced this replication, and 1,25(OH)₂D₃ had no affect in the absence of cells (Fig. 1).

IFN- and TNF are two of the best-characterized cytokine factors necessary for protection against TB (8, 42). The independence of 1,25(OH)₂D₃-mediated suppression of mycobacteria from these cytokines is evident in our experiments: both genes (together with the protective IL-12p40 gene) were strongly down-regulated by 1,25(OH)₂D₃ reflected by decreased cytokine secretion in culture (Figs. 4 and 3, respectively). Humans have only one intact IFN-inducible p47 GTPase (IRGC) whose expression has been reported from testis but not THP-1 cells (43). Although our data indicate that expression is present in primary myeloid cells, the effect of 1,25(OH)₂D₃ on IRGC expression was small and inconsistent, and so we cannot readily implicate this molecule in the effects we observed. 1,25(OH)₂D₃ did, however, increase the constitutive expression of NOS2A 3.9-fold in PBMC (Fig. 4E). NOS2A was not regulated by MTB, but at 24 h, its mean expression in MTB-stimulated PBMC was 5.25-fold increased (p = 0.005) by 1,25(OH)₂D₃. These effects are complex. NOS2A clearly appears 1,25(OH)₂D₃ inducible, and this gene is also known to be induced by IFN- (38). However, 1,25(OH)₂D₃ down-regulates IFN- (Figs. 3 and 4), and so the effects we observed in the PBMC of sensitized donors will have been composite. Unlike IFN- and TNF, we cannot therefore exclude a role for vitamin D-induced RNI in our system. However, the effects of inhibiting RNI formation on 1,25(OH)₂D₃-mediated suppression of BCG-lux were moderate (Fig. 5).

Cathelicidins are a structurally diverse family of antimicrobial peptide precursors with widespread distribution in mammals, characterized by the presence of a highly conserved cathelin domain of 100 residues. Humans express only one, hCAP-18, which is found in alveolar macrophages, lymphocytes, neutrophils, and epithelial cells (44). The promoter of the hCAP-18 gene contains a consensus vitamin D response element, and 1,25(OH)₂D₃ induces hCAP-18 gene expression in human cell lines (39). The protein product of hCAP-18 undergoes extracellular cleavage by the neutrophil azurophil granule proteinase 3 to generate a 37-residue peptide LL-37 (45). LL-37 also exerts immunomodulatory activity, being chemoattractant for MN, T cells, and neutrophils (46) and up-regulates IL-8 and MCP1 in human whole blood (47). It also possesses broad spectrum bactericidal activity: it kills microbes by disruption of the cell membrane (48). Therefore, it seemed plausible that 1,25(OH)₂D₃-induced antimycobacterial activity might be mediated by LL-37.

LL-37 (200 µg/ml) suppressed CFU to a similar extent as 10^–6 M 1,25(OH)₂D₃. The antimicrobial activity of LL-37 is known to be dependent on ionic environment (40). Our observation that a moderate concentration of LL-37 (5 µg/ml) induces superior suppression of H37Rv RLU and CFU under iron-limiting conditions supports the argument that LL-37 could be responsible for 1,25(OH)₂D₃-induced suppression of mycobacteria in MN (21). However, we did not detect LL-37 in the supernatant of 1,25(OH)₂D₃-stimulated PBMC by ELISA. Because immunostaining demonstrated the presence of intracellular hCAP-18 protein in monocytes (Fig. 6B), we attribute this finding either to a lack of secretion of hCAP-18 protein by PBMC or to a failure of extracellular cleavage of hCAP-18 protein to release LL-37 detectable by ELISA. It is feasible that neutrophils may fulfil the latter role in vivo.

Our demonstration that 1,25(OH)₂D₃-induced antimycobacterial activity is primarily mediated by nuclear-initiated signaling focused our investigation on transcriptional events. It is possible that the induction of LL-37 is a class effect and that other 1,25(OH)₂D₃-induced antimicrobial peptides may be of importance. The observation that antimycobacterial activity is primarily mediated via nuclear-initiated signaling may also have significance for drug development: doses of vitamin D administered to patients with active TB may be limited by induction of hypercalcemia (20). Therefore, there may be a place for the use of noncalcemic vitamin D analogs as novel adjunctive treatments for active TB. Our findings suggest that these analogs should possess activity at the nuclear VDR.

In conclusion, our data tend to support and extend the recent article (21) that vitamin D-inducible LL-37 may play a role in phagocyte defense against MTB. However, the effects of substantial concentrations of LL-37 on MTB are moderate, and the possibility that vitamin D may also regulate other antimicrobial peptides is a current focus of our work. Overall, our findings illuminate at the cellular level mechanisms by which micronutrient status might considerably influence susceptibility to TB. Greater research of these factors in clinical studies and by randomized controlled trials allied to in vitro research might delineate better the extent to which these factors may contribute to population susceptibility and novel routes to prevent and treat this complex and devastating disease.

	Acknowledgments

 
The funders of this study are public or nonprofit organizations that support science in general. They had no role in gathering, analyzing, or interpreting the data. We thank Bradley February for assistance in CFU enumeration and Drs. Geoff Packe and Mark Nicol for helpful discussions and review of the manuscript.

	Disclosures

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by the Wellcome Trust and the Department of Environmental Health, London Borough of Newham.

² Address correspondence and reprint requests to Dr. Robert Wilkinson, Imperial College London and University of Cape Town, Wernher and Beit South Building, Faculty of Health Science, Observatory, South Africa. E-mail address: r.j. wilkinson{at}imperial.ac.uk

³ Abbreviations used in this paper: TB, tuberculosis; 1,25(OH)₂D₃, 1,25 dihydroxyvitamin D₃; 25(OH)D₃, 25-hydroxyvitamin D₃; BCG, bacillus Calmette Guérin; L-NMMA, L-NG-monomethylarginine; LR, luminescence ratio; MN, monocyte; MOI, multiplicity of infection; PS, permeabilization solution; RLU, relative luciferase unit; RNI, reactive nitrogen intermediate; VDR, vitamin D receptor; VDR_mem, membrane-bound VDR; VDR_nuc, nuclear receptor VDR; 1-hydroxylase, 25-hydroxyvitamin D 1-hydroxylase.

Received for publication July 11, 2006. Accepted for publication March 13, 2007.

	References

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 

1. Corbett, E. L., B. Marston, G. J. Churchyard, K. M. De Cock. 2006. Tuberculosis in sub-Saharan Africa: opportunities, challenges, and change in the era of antiretroviral treatment. Lancet 367: 926-937. [Medline]
2. Espinal, M. A., A. Laszlo, L. Simonsen, F. Boulahbal, S. J. Kim, A. Reniero, S. Hoffner, H. L. Rieder, N. Binkin, C. Dye, et al 2001. Global trends in resistance to antituberculosis drugs: World Health Organization-International Union against Tuberculosis and Lung Disease Working Group on Anti-Tuberculosis Drug Resistance Surveillance. N. Engl. J. Med. 344: 1294-1303. [Abstract/Free Full Text]
3. Corbett, E. L., C. J. Watt, N. Walker, D. Maher, B. G. Williams, M. C. Raviglione, C. Dye. 2003. The growing burden of tuberculosis: global trends and interactions with the HIV epidemic. Arch. Intern. Med. 163: 1009-1021. [Abstract/Free Full Text]
4. Altare, F., A. Durandy, D. Lammas, J. F. Emile, S. Lamhamedi, F. Le Deist, P. Drysdale, E. Jouanguy, R. Doffinger, F. Bernaudin, et al 1998. Impairment of mycobacterial immunity in human interleukin-12 receptor deficiency. Science 280: 1432-1435. [Abstract/Free Full Text]
5. Keane, J., S. Gershon, R. P. Wise, E. Mirabile-Levens, J. Kasznica, W. D. Schwieterman, J. N. Siegel, M. M. Braun. 2001. Tuberculosis associated with infliximab, a tumor necrosis factor -neutralizing agent. N. Engl. J. Med. 345: 1098-1104. [Abstract/Free Full Text]
6. Newport, M. J., C. M. Huxley, S. Huston, C. M. Hawrylowicz, B. A. Oostra, R. Williamson, M. Levin. 1996. A mutation in the interferon -receptor gene and susceptibility to mycobacterial infection. N. Engl. J. Med. 335: 1941-1949. [Abstract/Free Full Text]
7. Kampmann, B., C. Hemingway, A. Stephens, R. Davidson, A. Goodsall, S. Anderson, M. Nicol, E. Scholvinck, D. Relman, S. Waddell, et al 2005. Acquired predisposition to mycobacterial disease due to autoantibodies to IFN-. J. Clin. Invest. 115: 2480-2488. [Medline]
8. Flynn, J. L., J. Chan. 2001. Immunology of tuberculosis. Annu. Rev. Immunol. 19: 93-129. [Medline]
9. Rose, A. M., J. M. Watson, C. Graham, A. J. Nunn, F. Drobniewski, L. P. Ormerod, J. H. Darbyshire, J. Leese. 2001. Tuberculosis at the end of the 20th century in England and Wales: results of a national survey in 1998. Thorax 56: 173-179. [Abstract/Free Full Text]
10. Talbot, E. A., M. Moore, E. McCray, N. J. Binkin. 2000. Tuberculosis among foreign-born persons in the United States, 1993–1998. J. Am. Med. Assoc. 284: 2894-2900. [Abstract/Free Full Text]
11. British Thoracic Society 2000. Control and prevention of tuberculosis in the United Kingdom: code of practice 2000: Joint Tuberculosis Committee of the British Thoracic Society. Thorax 55: 887-901. [Abstract/Free Full Text]
12. Trends in tuberculosis—United States, 2005. Morb. Mortal Wkly. Rep. 55: 2006305-308. [Medline]
13. Preece, M. A., S. Tomlinson, C. A. Ribot, J. Pietrek, H. T. Korn, D. M. Davies, J. A. Ford, M. G. Dunnigan, J. L. O’Riordan. 1975. Studies of vitamin D deficiency in man. Q. J. Med. 44: 575-589. [Medline]
14. Grenville-Mathers, R., J. B. Clark. 1979. The development of tuberculosis in Afro-Asian immigrants. Tubercle 60: 25-29. [Medline]
15. Wilkinson, R. J., M. Llewelyn, Z. Toossi, P. Patel, G. Pasvol, A. Lalvani, D. Wright, M. Latif, R. N. Davidson. 2000. Influence of vitamin D deficiency and vitamin D receptor polymorphisms on susceptibility to tuberculosis amongst Gujarati Asians in west London: a case-control study. Lancet 355: 618-621. [Medline]
16. Ustianowski, A., R. Shaffer, S. Collin, R. J. Wilkinson, R. N. Davidson. 2005. Prevalence and associations of vitamin D deficiency in foreign-born persons with tuberculosis in London. J. Infect. 50: 432-437. [Medline]
17. Crowle, A., E. Ross, M. May. 1987. Inhibition by 1,25(OH)₂-vitamin D₃ of the multiplication of virulent tubercle bacilli in cultured human macrophages. Infect. Immun. 55: 2945-2950. [Abstract/Free Full Text]
18. Davies, P., R. Brown, J. Woodhead. 1985. Serum concentrations of vitamin D metabolites in untreated tuberculosis. Thorax 40: 187-190. [Abstract/Free Full Text]
19. Bellamy, R., C. Ruwende, T. Corrah, K. P. McAdam, M. Thursz, H. C. Whittle, A. V. Hill. 1999. Tuberculosis and chronic hepatitis B virus infection in Africans and variation in the vitamin D receptor gene. J. Infect. Dis. 179: 721-724. [Medline]
20. Martineau, A. R., F. U. Honecker, R. J. Wilkinson, C. J. Griffiths. 2007. Vitamin D in the treatment of pulmonary tuberculosis. J. Steroid Biochem. Mol. Biol. 103: 793-798. [Medline]
21. Liu, P. T., S. Stenger, H. Li, L. Wenzel, B. H. Tan, S. Krutzik, M. T. Ochoa, J. Schauber, K. Wu, C. Meinken, et al 2006. Toll-like receptor triggering of a vitamin D-mediated human antimicrobial response. Science 311: 1770-1773. [Abstract/Free Full Text]
22. Stoffels, K., L. Overbergh, A. Giulietti, L. Verlinden, R. Bouillon, C. Mathieu. 2006. Immune regulation of 25-hydroxyvitamin-D₃-1-hydroxylase in human monocytes. J. Bone Miner. Res. 21: 37-47. [Medline]
23. Vidal, M., C. V. Ramana, A. S. Dusso. 2002. Stat1-vitamin D receptor interactions antagonize 1,25-dihydroxyvitamin D transcriptional activity and enhance stat1-mediated transcription. Mol. Cell. Biol. 22: 2777-2787. [Abstract/Free Full Text]
24. Rook, G. A., J. Steele, L. Fraher, S. Barker, R. Karmali, J. O’Riordan, J. Stanford. 1986. Vitamin D₃, interferon, and control of proliferation of Mycobacterium tuberculosis by human monocytes. Immunology 57: 159-163. [Medline]
25. Sly, L. M., M. Lopez, W. M. Nauseef, N. E. Reiner. 2001. 1,25-Dihydroxyvitamin D₃-induced monocyte antimycobacterial activity is regulated by phosphatidylinositol 3-kinase and mediated by the NADPH-dependent phagocyte oxidase. J. Biol. Chem. 276: 35482-35493. [Abstract/Free Full Text]
26. Hmama, Z., K. Sendide, A. Talal, R. Garcia, K. Dobos, N. E. Reiner. 2004. Quantitative analysis of phagolysosome fusion in intact cells: inhibition by mycobacterial lipoarabinomannan and rescue by an 1,25-dihydroxyvitamin D₃-phosphoinositide 3-kinase pathway. J. Cell Sci. 117: 2131-2140. [Abstract/Free Full Text]
27. Norman, A. W., W. H. Okamura, J. E. Bishop, H. L. Henry. 2002. Update on biological actions of 1,25(OH)₂-vitamin D₃ (rapid effects) and 24R,25(OH)₂-vitamin D₃. Mol. Cell. Endocrinol. 197: 1-13. [Medline]
28. Rockett, K. A., R. Brookes, I. Udalova, V. Vidal, A. V. Hill, D. Kwiatkowski. 1998. 1,25-Dihydroxyvitamin D₃ induces nitric oxide synthase and suppresses growth of Mycobacterium tuberculosis in a human macrophage-like cell line. Infect. Immun. 66: 5314-5321. [Abstract/Free Full Text]
29. MacMicking, J. D., G. A. Taylor, J. D. McKinney. 2003. Immune control of tuberculosis by IFN--inducible LRG-47. Science 302: 654-659. [Abstract/Free Full Text]
30. Stewart, G. R., K. A. Wilkinson, S. M. Newton, S. M. Sullivan, O. Neyrolles, J. R. Wain, J. Patel, K. L. Pool, D. B. Young, R. J. Wilkinson. 2005. Effect of deletion or overexpression of the 19-kilodalton lipoprotein Rv3763 on the innate response to Mycobacterium tuberculosis. Infect. Immun. 73: 6831-6837. [Abstract/Free Full Text]
31. Turner, D. J., S. L. Hoyle, V. A. Snewin, M. P. Gares, I. N. Brown, D. B. Young. 2002. An ex vivo culture model for screening drug activity against in vivo phenotypes of Mycobacterium tuberculosis. Microbiology 148: 2929-2936. [Abstract/Free Full Text]
32. Snewin, V. A., M. P. Gares, P. O. Gaora, Z. Hasan, I. N. Brown, D. B. Young. 1999. Assessment of immunity to mycobacterial infection with luciferase reporter constructs. Infect. Immun. 67: 4586-4593. [Abstract/Free Full Text]
33. Kampmann, B., P. O. Gaora, V. A. Snewin, M. P. Gares, D. B. Young, M. Levin. 2000. Evaluation of human antimycobacterial immunity using recombinant reporter mycobacteria. J. Infect. Dis. 182: 895-901. [Medline]
34. Ratledge, C.. 2004. Iron, mycobacteria and tuberculosis. Tuberculosis 84: 110-130. [Medline]
35. Wilkinson, R. J., L. E. DesJardin, N. Islam, B. M. Gibson, R. A. Kanost, K. A. Wilkinson, D. Poelman, K. D. Eisenach, Z. Toossi. 2001. An increase in expression of a M. tuberculosis mycolyl transferase gene (fbpB) occurs early after infection of human monocytes. Mol. Microbiol. 39: 813-821. [Medline]
36. Sorensen, O., J. B. Cowland, J. Askaa, N. Borregaard. 1997. An ELISA for hCAP-18, the cathelicidin present in human neutrophils and plasma. J. Immunol. Methods 206: 53-59. [Medline]
37. Sorensen, O., T. Bratt, A. H. Johnsen, M. T. Madsen, N. Borregaard. 1999. The human antibacterial cathelicidin, hCAP-18, is bound to lipoproteins in plasma. J. Biol. Chem. 274: 22445-22451. [Abstract/Free Full Text]
38. MacMicking, J. D., R. J. North, R. LaCourse, J. S. Mudgett, S. K. Shah, C. F. Nathan. 1997. Identification of nitric oxide synthase as a protective locus against tuberculosis. Proc. Natl. Acad. Sci. USA 94: 5243-5248. [Abstract/Free Full Text]
39. Wang, T. T., F. P. Nestel, V. Bourdeau, Y. Nagai, Q. Wang, J. Liao, L. Tavera-Mendoza, R. Lin, J. W. Hanrahan, S. Mader, J. H. White. 2004. Cutting edge: 1,25-dihydroxyvitamin D₃ is a direct inducer of antimicrobial peptide gene expression. J. Immunol. 173: 2909-2912. [Abstract/Free Full Text]
40. Dorschner, R. A., B. Lopez-Garcia, A. Peschel, D. Kraus, K. Morikawa, V. Nizet, R. L. Gallo. 2006. The mammalian ionic environment dictates microbial susceptibility to antimicrobial defense peptides. FASEB J. 20: 35-42. [Abstract/Free Full Text]
41. Wilkinson, K. A., G. R. Stewart, S. M. Newton, H. M. Vordermeier, J. R. Wain, H. N. Murphy, K. Horner, D. B. Young, R. J. Wilkinson. 2005. Infection biology of a novel -crystallin of Mycobacterium tuberculosis: Acr2. J. Immunol. 174: 4237-4243. [Abstract/Free Full Text]
42. Wilkinson, R. J., D. B. Young. 2004. Novel vaccines against tuberculosis. M. Levine, and J. Kaper, and R. Rappuoli, and M. Liu, and M. Good, eds. New Generation Vaccines 3rd Ed.519-535. Marcel Dekker, New York.
43. Bekpen, C., J. P. Hunn, C. Rohde, I. Parvanova, L. Guethlein, D. M. Dunn, E. Glowalla, M. Leptin, J. C. Howard. 2005. The interferon-inducible p47 (IRG) GTPases in vertebrates: loss of the cell autonomous resistance mechanism in the human lineage. Genome Biol. 6: R92[Medline]
44. Wah, J., A. Wellek, M. Frankenberger, P. Unterberger, U. Welsch, R. Bals. 2006. Antimicrobial peptides are present in immune and host defense cells of the human respiratory and gastrointestinal tracts. Cell Tissue Res. 324: 449-456. [Medline]
45. Sorensen, O. E., P. Follin, A. H. Johnsen, J. Calafat, G. S. Tjabringa, P. S. Hiemstra, N. Borregaard. 2001. Human cathelicidin, hCAP-18, is processed to the antimicrobial peptide LL-37 by extracellular cleavage with proteinase 3. Blood 97: 3951-3959. [Abstract/Free Full Text]
46. De, Y., Q. Chen, A. P. Schmidt, G. M. Anderson, J. M. Wang, J. Wooters, J. J. Oppenheim, O. Chertov. 2000. LL-37, the neutrophil granule- and epithelial cell-derived cathelicidin, utilizes formyl peptide receptor-like 1 (FPRL1) as a receptor to chemoattract human peripheral blood neutrophils, monocytes, and T cells. J. Exp. Med. 192: 1069-1074. [Abstract/Free Full Text]
47. Scott, M. G., D. J. Davidson, M. R. Gold, D. Bowdish, R. E. Hancock. 2002. The human antimicrobial peptide LL-37 is a multifunctional modulator of innate immune responses. J. Immunol. 169: 3883-3891. [Abstract/Free Full Text]
48. Henzler-Wildman, K. A., G. V. Martinez, M. F. Brown, A. Ramamoorthy. 2004. Perturbation of the hydrophobic core of lipid bilayers by the human antimicrobial peptide LL-37. Biochemistry 43: 8459-8469. [Medline]
49. Norman, A. W., W. H. Okamura, M. W. Hammond, J. E. Bishop, M. C. Dormanen, R. Bouillon, H. van Baelen, A. L. Ridall, E. Daane, R. Khoury, M. C. Farach-Carson. 1997. Comparison of 6-s-cis- and 6-s-trans-locked analogs of 1,25-dihydroxyvitamin D₃ indicates that the 6-s-cis conformation is preferred for rapid nongenomic biological responses and that neither 6-s-cis- nor 6-s-trans-locked analogs are preferred for genomic biological responses. Mol. Endocrinol. 11: 1518-1531. [Abstract/Free Full Text]
50. Zhou, L. X., I. Nemere, A. W. Norman. 1992. 1,25-Dihydroxyvitamin D₃ analog structure-function assessment of the rapid stimulation of intestinal calcium absorption (transcaltachia). J. Bone Miner. Res. 7: 457-463. [Medline]
51. Bula, C. M., J. E. Bishop, S. Ishizuka, A. W. Norman. 2000. 25-Dehydro-1-hydroxyvitamin D3-26,23S-lactone antagonizes the nuclear vitamin D receptor by mediating a unique noncovalent conformational change. Mol. Endocrinol. 14: 1788-1796. [Abstract/Free Full Text]
52. Norman, A. W., R. Bouillon, M. C. Farach-Carson, J. E. Bishop, L. X. Zhou, I. Nemere, J. Zhao, K. R. Muralidharan, W. H. Okamura. 1993. Demonstration that 1,25-dihydroxyvitamin D₃ is an antagonist of the nongenomic but not genomic biological responses and biological profile of the three A-ring diastereomers of 1,25-dihydroxyvitamin D₃. J. Biol. Chem. 268: 20022-20030. [Abstract/Free Full Text]

This article has been cited by other articles:

				R. J. Wilkinson and C. Lange Vitamin D and Tuberculosis: New Light on a Potent Biologic Therapy? Am. J. Respir. Crit. Care Med., May 1, 2009; 179(9): 740 - 742. [Full Text] [PDF]

				J. S. Adams, S. Ren, P. T. Liu, R. F. Chun, V. Lagishetty, A. F. Gombart, N. Borregaard, R. L. Modlin, and M. Hewison Vitamin D-Directed Rheostatic Regulation of Monocyte Antibacterial Responses J. Immunol., April 1, 2009; 182(7): 4289 - 4295. [Abstract] [Full Text] [PDF]

				C.-S. Yang, D.-M. Shin, K.-H. Kim, Z.-W. Lee, C.-H. Lee, S. G. Park, Y. S. Bae, and E.-K. Jo NADPH Oxidase 2 Interaction with TLR2 Is Required for Efficient Innate Immune Responses to Mycobacteria via Cathelicidin Expression J. Immunol., March 15, 2009; 182(6): 3696 - 3705. [Abstract] [Full Text] [PDF]

				N. Liu, A.T. Kaplan, J. Low, L. Nguyen, G.Y. Liu, O. Equils, and M. Hewison Vitamin D Induces Innate Antibacterial Responses in Human Trophoblasts via an Intracrine Pathway Biol Reprod, March 1, 2009; 80(3): 398 - 406. [Abstract] [Full Text] [PDF]

				S. R. Krutzik, M. Hewison, P. T. Liu, J. A. Robles, S. Stenger, J. S. Adams, and R. L. Modlin IL-15 Links TLR2/1-Induced Macrophage Differentiation to the Vitamin D-Dependent Antimicrobial Pathway J. Immunol., November 15, 2008; 181(10): 7115 - 7120. [Abstract] [Full Text] [PDF]

				R. Bouillon, G. Carmeliet, L. Verlinden, E. van Etten, A. Verstuyf, H. F. Luderer, L. Lieben, C. Mathieu, and M. Demay Vitamin D and Human Health: Lessons from Vitamin D Receptor Null Mice Endocr. Rev., October 1, 2008; 29(6): 726 - 776. [Abstract] [Full Text] [PDF]

				N. Liu, L. Nguyen, R. F. Chun, V. Lagishetty, S. Ren, S. Wu, B. Hollis, H. F. DeLuca, J. S. Adams, and M. Hewison Altered Endocrine and Autocrine Metabolism of Vitamin D in a Mouse Model of Gastrointestinal Inflammation Endocrinology, October 1, 2008; 149(10): 4799 - 4808. [Abstract] [Full Text] [PDF]

				R. F Chun, J. S Adams, and M. Hewison Back to the future: a new look at 'old' vitamin D J. Endocrinol., August 1, 2008; 198(2): 261 - 269. [Abstract] [Full Text] [PDF]

				W. W. Yew and C. C. Leung Update in Tuberculosis 2007 Am. J. Respir. Crit. Care Med., March 1, 2008; 177(5): 479 - 485. [Full Text] [PDF]

				M. Zasloff Antimicrobial Peptides, Innate Immunity, and the Normally Sterile Urinary Tract J. Am. Soc. Nephrol., November 1, 2007; 18(11): 2810 - 2816. [Abstract] [Full Text] [PDF]

				A. L. Jones, R. H. Mertz, D. J. Carl, and C. E. Rubens A Streptococcal Penicillin-Binding Protein Is Critical for Resisting Innate Airway Defenses in the Neonatal Lung J. Immunol., September 1, 2007; 179(5): 3196 - 3202. [Abstract] [Full Text] [PDF]

				P. T. Liu, S. Stenger, D. H. Tang, and R. L. Modlin Cutting Edge: Vitamin D-Mediated Human Antimicrobial Activity against Mycobacterium tuberculosis Is Dependent on the Induction of Cathelicidin J. Immunol., August 15, 2007; 179(4): 2060 - 2063. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) A correction has been published Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Martineau, A. R. Articles by Wilkinson, R. J. Search for Related Content PubMed PubMed Citation Articles by Martineau, A. R. Articles by Wilkinson, R. J.